Primer, probe and kit for detecting mycobacterium tuberculosis based on reference gene and bacterial gene

Abstract

The invention relates to the technical field of pathogenic bacteria detection, and discloses a primer, a probe and a kit for detecting mycobacterium tuberculosis based on a reference gene and a bacterial gene. According to the technical scheme, an absolute quantitative qPCR technology is optimized and innovated, and a double-fluorescent probe is designed and is used for respectively marking a specific gene and the reference gene of Mtb; and simultaneously determining the copy number of bacterial genes and the copy number of reference genes in the Mtb and macrophage co-culture system by using an absolute quantitative qPCR (quantitative polymerase chain reaction) technology. The optimized technology not only has innovativeness, but also can be used as a direct index for evaluating the anti-tuberculosis effect, and has wide popularization and application prospects in scientific research and even clinical diagnosis of tuberculosis treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, and in particular to a primer, a probe and a kit for detecting Mycobacterium tuberculosis based on an internal reference gene and a bacterial gene. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, which mainly affects the lungs, but can also invade other organs such as lymph nodes, bone marrow, meninges, etc. Tuberculosis is one of the major public health problems facing the world and the main source of death among adults. In recent years, the incidence of tuberculosis has tended to rise again due to large-scale population mobility and the development of drug resistance of the bacteria themselves.

[0003] Mycobacterium tuberculosis (Mtb) is the pathogen of tuberculosis. Currently, the diagnosis of Mtb mainly adopts a method combining laboratory diagnosis with clinical signs. Laboratory diagnosis mainly includes smear staining, bacterial culture, antigen-antibody detection, PCR and other methods. Among them, bacterial culture of patient tissue or body fluid specimens is currently the gold standard for detecting Mycobacterium tuberculosis, but its disadvantage is that the detection cycle is long (requires 8 weeks) and it is difficult to achieve rapid detection of pathogenic bacteria. Therefore, it is easy to delay the condition in clinical practice and is not conducive to early treatment. Antigen and antibody detection usually uses ELISA (enzyme-linked immunosorbent assay), which has the advantages of simple operation and low price, but the detection specificity and sensitivity need to be improved. With the development of molecular biology, absolute quantitative qPCR (Quantitative Polymerase Chain Reaction) technology has become a common experimental method and is widely used in pathogen detection, gene expression analysis and other fields. However, when using this technology to detect pathogens such as Mycobacterium tuberculosis (Mtb), there are still some significant challenges and limitations:

[0004] 1. Sample processing and purification: Mycobacterium tuberculosis is often present in complex biological samples, such as sputum, blood, tissue, etc. These samples contain a large number of host cells and other microorganisms, which increases the difficulty of extracting high-quality template DNA / RNA. In addition, Mycobacterium tuberculosis has a thick and tough cell wall, and conventional lysis methods are difficult to effectively destroy its structure, resulting in incomplete release of nucleic acids, which in turn affects the detection sensitivity. Moreover, certain components in the sample (such as hemoglobin, bile salts, polysaccharides, etc.) may inhibit the PCR reaction and reduce the amplification efficiency. Although the impact of such substances can be reduced by optimizing the purification steps, it is still challenging to completely eliminate them.

[0005] 2. Detection sensitivity and specificity issues: In latent infection or early active tuberculosis cases, the number of Mycobacterium tuberculosis is often very low, which places higher sensitivity requirements on qPCR detection. However, increasing sensitivity may also introduce more false positive signals. In addition, highly conservative primer combinations designed to increase sensitivity may simultaneously amplify DNA / RNA of other related species, causing misjudgment. Therefore, how to ensure high specificity while ensuring sufficient sensitivity is an urgent problem to be solved. Summary of the invention

[0006] The present invention aims to provide a primer, a probe and a kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes, aiming to take into account the sensitivity and specificity of Mycobacterium tuberculosis load detection while improving the convenience of operation.

[0007] To achieve the above-mentioned object, the present invention adopts the following technical scheme: a primer for detecting Mycobacterium tuberculosis based on an internal reference gene and a bacterial gene, the primer comprising a bacteria-specific insertion sequence IS986 primer and an internal reference gene β-actin primer, the nucleotide sequence of the bacteria-specific insertion sequence IS986 primer is shown in SEQ ID NO: 1, and the nucleotide sequences of the internal reference gene β-actin primer are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0008] Preferably, as an improvement, a probe for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes comprises a bacteria-specific insertion sequence IS986 probe and an internal reference gene β-actin probe, the nucleotide sequence of the bacteria-specific insertion sequence IS986 probe is shown in SEQ ID NO:4, and the nucleotide sequence of the internal reference gene β-actin probe is shown in SEQ ID NO:5 and SEQ ID NO:6.

[0009] Preferably, as an improvement, the 5' end of the probe is labeled with a fluorescent group, the 3' end of the probe is labeled with a quenching group, the fluorescent group of the internal reference gene β-actin probe is red fluorescent ROX, the fluorescent group of the bacteria-specific insertion sequence IS986 probe is green fluorescent FAM; the quenching group is BHQ.

[0010] Preferably, as an improvement, a kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes comprises the primers and probes described above.

[0011] In the present technical scheme, when preparing a kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes, in addition to conventional real-time fluorescence quantitative PCR reagents (including: Taq enzyme, dNTP reagent, PCR buffer, sterile water), it also includes the above-mentioned internal reference genes, primers and probes of bacterial genes.

[0012] Preferably, as an improvement, the total DNA in the Mtb and macrophage co-culture system is extracted before use, and the extraction method is a glass bead method.

[0013] Preferably, as an improvement, when the kit is used to detect the Mtb load, the Mtb load = the Mtb IS986 bacterial gene copy number / the internal reference gene β-actin copy number.

[0014] Preferably, as an improvement, the technical formula for copy number is copy number (copies / μL) = DNA concentration (ng / μL) × 6.02 × 10 23 ×10 -9 / DNA relative molecular mass, where DNA relative molecular mass = number of base pairs in DNA fragment × 660.

[0015] Preferably, as an improvement, the Tm of the gradient PCR reaction of primers for the bacteria-specific insertion sequence IS986 in the co-culture system of Mtb and macrophages is 52.5-64°C.

[0016] The principle and advantage of this scheme are: in actual application, in this technical scheme, in order to provide an evaluation reference for measuring the effect of anti-tuberculosis drugs and the therapeutic effect, the determination method of intracellular Mycobacterium tuberculosis (Mtb) load is optimized. On the basis of absolute quantitative qPCR technology (probe method), different fluorescently labeled internal reference gene probes and bacterial gene probes are used to simplify the experimental operation, and the copy number of the internal reference gene and the bacterial gene can be detected in the same reaction system at the same time, and the formula: Mtb load = Mtb IS986 gene copy number / internal reference gene β-actin copy number is used for calculation, and the result is the Mtb load per unit cell. Compared with the traditional method, two reaction systems need to be prepared, one system detects the copy number of the internal reference gene, and the other system detects the copy number of the bacterial gene, and the same template DNA is added respectively, the template DNA of this technical scheme only needs to be added once, which reduces the error of repeated addition and makes the operation easier.

[0017] In addition, in the present technical solution, primers with higher specificity are designed for different bacterial specific insertion sequences IS986. Compared with the primers designed for the existing bacterial insertion sequence IS6110 (GenBank: X17348.1), the primers designed for the bacterial insertion sequence IS986 have higher specificity. The essential difference between the bacterial specific insertion sequence IS986 and the bacterial insertion sequence IS6110 lies in: molecular weight and base sequence. The primers and probes designed for the bacterial insertion sequence IS986 (GenBank: X52471.1) and the human and mouse internal reference gene β-actin, both pairs of primers can amplify characteristic bands with the correct molecular weight, and the Mtb specific insertion sequence IS986 can only amplify specific products of the correct size in Mtb-infected samples, while no specific products appear in PBS-treated cell samples.

[0018] Through cell experiments: Mtb infected macrophages from humans and mice at different time points, the optimized Mtb load detection method was able to accurately measure the intracellular Mtb load in Mtb-infected humans (THP-1 induced into macrophages by phorbol ester) and mouse macrophages (RAW264.7).

[0019] During the technical research and development period, the total DNA extraction in the co-culture system of Mtb and cells was one of the difficulties in the development of this technical solution: because Mycobacterium tuberculosis has a thick cell wall and a high lipid content, the effect of extracting the bacterial DNA using a conventional bacterial DNA extraction kit is not good. This solution compared the concentration and quality of total DNA extracted by the Trizol method and the glass bead method, and finally found that the quality of total DNA extracted by the glass bead method was higher and was conducive to the subsequent determination of Mtb load. In addition, the exploration of the amplification conditions of the primers designed for the bacterial specific insertion sequences IS986 and IS6110 is another research and development difficulty of this technical solution: including the selection of annealing temperature Tm and primer specificity. Finally, preliminary experiments were carried out in Mtb and human macrophages, Mtb and mouse macrophages to detect the Mtb load. The results showed that there were amplification products only in the reaction system with bacterial DNA, and there was no non-specific amplification; and the bands of the amplification products were uniform and bright; representing that this solution has high concentration and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow chart of the absolute quantitative qPCR technology used in an embodiment of the present invention to determine the Mtb load in Mtb-infected cells.

[0021] Figure 2 This is a graph showing the results of gradient PCR products of Mtb specific insertion sequences IS986 and IS6110 detected by agarose gel electrophoresis in an embodiment of the present invention.

[0022] Figure 3 This is a graph showing the results of agarose gel electrophoresis detection of PCR products of Mtb-specific insertion sequence IS986 and mouse internal reference gene β-actin in an embodiment of the present invention.

[0023] Figure 4 This is a graph showing the results of agarose gel electrophoresis detection of PCR amplification products of the newly designed human internal reference gene β-actin in an embodiment of the present invention.

[0024] Figure 5 This is the standard curve of the Mycobacterium tuberculosis specific insertion sequence IS986 in the examples of the present invention.

[0025] Figure 6 This is the standard curve of the human / mouse internal reference gene β-actin in the embodiments of the present invention.

[0026] Figure 7 This is a graph showing the absolute quantitative detection result of the Mtb load in Mtb-infected THP-1 / RAW264.7 macrophages by the Taqman probe method in an embodiment of the present invention.

[0027] Figure 8 This is a schematic diagram of the total DNA extracted by the Trizol method using RNase-Free water in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0029] Example 1

[0030] like Figure 1 As shown: The detection method of the Mtb load in Mtb-infected cells using absolute quantitative qPCR technology includes the following steps:

[0031] Step 1: Prepare a standard with known concentration;

[0032] Step 2: Draw a standard curve;

[0033] Step 3: Determine the gene copy number, Mtb load = Mtb IS986 gene copy number / reference gene β-actin copy number.

[0034] Experimental Example 1 Study on the specificity of primers designed for different bacterial genes

[0035] 1. Selection of Mycobacterium tuberculosis-specific primers and annealing temperature

[0036] Using the extracted total DNA as a template, including the PBS-treated control cell group and the Mtb-infected experimental cell group, gradient PCR amplification was performed using primers targeting the tuberculosis-specific insertion sequence IS986 and IS6110 to select the appropriate annealing temperature and primers with better specificity. Among them, the molecular weight of the amplification product of the IS986 primer is 131bp, while the molecular weight of the amplification product of the IS6110 primer is 83bp. The primer and probe sequences are shown in Table 1:

[0037] Table 1

[0038]

[0039] The design conditions of the gradient PCR amplification system are shown in Table 2-4:

[0040] Table 2 Amplification system of target fragments

[0041]

[0042] Table 3 Amplification conditions of target fragments

[0043]

[0044] Note: *Tm is 64°C.

[0045] Table 4 Amplification conditions of target fragment gradient PCR

[0046]

[0047] *The Tm range of gradient PCR reaction with primers IS986 and IS6110 in the co-culture system of mouse macrophages RAW264.7 and Mtb is 52.5-64℃.

[0048] Mouse macrophage RAW264.7 infected with Mtb was used as the experimental group, and the same type of cells treated with an equal volume of PBS were used as the control group. After 72 hours, the cell samples of each group were collected, and the total DNA in the experimental group and the control group cells was extracted respectively. The total DNA extraction method adopted the glass bead method. Different PCR reaction systems were constructed using primers of Mtb-specific insertion sequences IS986 and IS6110, respectively, with the total DNA in the experimental group cells and the total DNA in the control group cells as templates. The reagents and volumes added to the reaction system are shown in Table 2, and the amplification conditions of the gradient PCR are shown in Table 3-4. After the PCR reaction was completed, the product was detected by 2% agarose gel electrophoresis, and the agarose gel was developed with a gel imager.

[0049] The results are as follows Figure 2As shown, the annealing temperature ranged from 52.5°C to 64°C, with a total of 8 gradients. A comparative analysis was conducted from the aspects of amplification effect and primer specificity, and it was found that the primers targeting the insertion sequence IS986 were superior to the primers targeting the insertion element IS6110. Therefore, in subsequent experiments, primers for amplifying Mtb were selected from IS986, and the annealing temperature was selected to be 64°C (as shown in the red box). Only at an annealing temperature of 64°C can the specific products of bacteria be correctly amplified in the Mtb-infected experimental group, and no non-specific products appear in the PBS control group. (Nucleic acid molecular weight standard: DL 2000; red arrow: specific amplification product of Mycobacterium tuberculosis; black arrow: primer dimer)

[0050] Experimental Example 2: Validation of primers and probes designed for bacterial insertion sequence IS986 (GenBank: X52471.1) and human and mouse internal reference gene β-actin

[0051] The primer specificity and annealing temperature of the reference gene β-actin and the Mtb-specific insertion sequence IS986 were detected using the mouse macrophage RAW264.7 sample infected with Mtb (PBS-treated as the control group). The reagents and volumes added to the reaction system are shown in Table 2, and the PCR amplification conditions are shown in Table 3. The results are shown in Figure 3 As shown, both pairs of primers can amplify characteristic bands with the correct molecular size, and the Mtb-specific insertion sequence IS986 can only amplify specific products with the correct size in Mtb-infected samples, while no specific products appear in PBS-treated cell samples (nucleic acid molecular weight standard: DL 500, annealing temperature 64°C).

[0052] The primer specificity and annealing temperature of the reference gene β-actin and the Mtb-specific insertion sequence IS986 were detected using human THP-1 cell samples infected with Mtb (PBS-treated as the control group). The reagents and volumes added to the reaction system are shown in Table 2, and the PCR amplification conditions are shown in Table 3. The results are shown in Figure 4 As shown, both pairs of primers can amplify characteristic bands with the correct molecular size, and the Mtb-specific insertion sequence IS986 can only amplify specific products with the correct size in Mtb-infected samples, while no specific products appear in PBS-treated cell samples (nucleic acid molecular weight standard: DL 500, annealing temperature 67.3°C).

[0053] 1. Preparation of absolute quantitative standards for tuberculosis TaqMan probe qPCR

[0054] The total DNA of the experimental cell group infected with Mtb was extracted as a template for the PCR reaction, and PCR amplification was performed using primers targeting the tuberculosis-specific insertion sequence IS986. After the PCR reaction was completed, the product was subjected to nucleic acid electrophoresis to test the purity of the product.

[0055] If the size of the PCR amplification product is consistent and the purity is good, the DNA concentration of the PCR product is detected by NanoDrop 2000 micro-nucleic acid protein detector. The copy number of the target fragment is calculated based on the measured PCR product DNA concentration. The calculation formula is as follows:

[0056] Copy number (copies / μL) = DNA concentration (ng / μL) × 6.02 × 10 23 ×10 -9 / DNA relative molecular mass

[0057] DNA molecular mass = number of base pairs in DNA fragment × 660

[0058] The PCR amplification product with a known copy number is used as the standard for the next step of the tuberculosis experiment. The standard is diluted 10 times in series with the sample diluent EASY Dilution, and diluted to 8-10 concentration gradients to cover the entire concentration range of the corresponding target gene in the sample to be tested.

[0059] 2. Check the quality of the absolute quantitative standard of qPCR by TaqMan probe method for tuberculosis

[0060] The diluted standard was used as a template for TaqMan Real-time PCR amplification, and the quality and dilution effect of the standard were finally tested, and a standard curve was drawn. The TaqMan Real-time PCR reaction system is shown in Table 5.

[0061] Table 5 TaqMan Real-time PCR reaction system

[0062]

[0063] TaqMan Real-time PCR amplification conditions are shown in Table 6:

[0064] Table 6 TaqMan Real-time PCR amplification conditions

[0065]

[0066]

[0067] *The Tm of IS986 and β-actin primers in the co-culture system of mouse macrophages RAW264.7 and Mtb is 64℃, and the Tm of IS986 and β-actin primers in the co-culture system of human cells THP-1 and Mtb is 67.3℃.

[0068] When the amplification results show the correlation with the standard curve (R 2 ) is greater than 98% and the amplification efficiency (E) is between 90% and 110%, indicating that the standard is of good quality and can be used for subsequent real-time PCR absolute quantification experiments.

[0069] 3. Produce human and mouse β-actin gene TaqMan probe qPCR absolute quantitative standards

[0070] The extracted total DNA was used as a template and PCR amplification was performed using primers targeting the human / mouse internal reference gene β-actin. The molecular weight of the amplified product of the human β-actin primer is 211bp, while the molecular weight of the amplified product of the mouse β-actin primer is 185bp. The PCR reaction system is shown in Table 2, and the amplification conditions are shown in Table 3. The DNA concentration of the PCR product was detected using the NanoDrop 2000 micro-nucleic acid protein detector. The copy number of the target fragment was calculated based on the measured concentration of the PCR product DNA. The calculation formula is as follows:

[0071] Copy number (copies / μL) = DNA concentration (ng / μL) × 6.02 × 10 23 ×10 -9 / DNA relative molecular mass

[0072] DNA molecular mass = number of base pairs in DNA fragment × 660

[0073] The PCR amplification product with a known copy number is used as the standard for the next step of the tuberculosis experiment. The standard is diluted 10 times in series with the sample diluent EASY Dilution, and diluted to 8-10 concentration gradients to cover the entire concentration range of the corresponding target gene in the sample to be tested.

[0074] 4. Testing of human and mouse β-actin gene TaqMan probe qPCR absolute quantitative standards

[0075] The diluted standard was used as a template for TaqMan Real-time PCR amplification, and the quality of the standard and the dilution effect were finally tested, and a standard curve was drawn. The TaqMan Real-time PCR reaction system is shown in Table 5, and the amplification conditions are shown in Table 6.

[0076] When the amplification results show the correlation with the standard curve (R2 ) is greater than 98% and the amplification efficiency (E) is between 90% and 110%, indicating that the standard is of good quality and can be used for subsequent real-time PCR absolute quantification experiments.

[0077] Experimental results: Successful preparation of the absolute quantitative standard of the tuberculosis-specific insertion sequence IS986

[0078] Human monocytic leukemia THP-1 cells were induced into macrophages by PMA and then infected with Mtb. The total DNA of bacteria and cells was extracted as a template to amplify the Mtb-specific insertion sequence IS986. The DNA concentration was measured by NanoDrop 2000 micro-nucleic acid protein detector to be 891.6 ng / μL. The copy number of the amplified product was calculated to be 6.21×10 12 The standard was diluted 10 times in series, with a total of 7 concentrations. The diluted standard was used as a template, and TaqMan Real-time PCR amplification was performed with IS986 specific primers and probes to draw a standard curve, such as Figure 5 A: R of the standard curve of the Mtb-specific insertion sequence IS986 in the co-culture system of human macrophages and Mtb. 2 The value is 0.9955, the slope is -3.214, and the amplification efficiency is calculated according to the formula E=-1+10(-1 / slope)=104.71%.

[0079] After infecting mouse mononuclear macrophage leukemia cells RAW264.7 with Mtb, the total DNA of bacteria and cells was extracted as a template to amplify the Mtb-specific insertion sequence IS986. The DNA concentration was measured by NanoDrop 2000 micro-nucleic acid protein detector to be 803.6 ng / μL. The copy number of the amplified product was calculated to be 5.6×10 12 The standard was diluted 10 times in series, with a total of 8 concentrations. The diluted standard was used as a template, and the IS986 specific primers and probe were used for TaqMan Real-time PCR amplification to draw a standard curve, such as Figure 5 Figure B: R of the standard curve of the Mtb-specific insertion sequence IS986 in the co-culture system of mouse macrophages and Mtb. 2 The value is 0.9969, the slope is -3.2741, and the amplification efficiency is calculated according to the formula E=-1+10(-1 / slope)=102.04%.

[0080] The amplification efficiency E of the two standard curves was in the range of 90-110%, and R 2They were also greater than 0.98, indicating that the quality of the standards was good and the standard curve had high credibility, and could be used for subsequent Real-time PCR absolute quantification experiments.

[0081] Experimental results: Successfully prepared the absolute quantitative standard of human / mouse internal reference gene β-actin

[0082] After human monocytic leukemia THP-1 cells were induced into macrophages by PMA, they were treated with PBS and the total cell DNA was extracted as a template to amplify the human internal reference gene β-actin. The DNA concentration was measured by NanoDrop 2000 micro-nucleic acid protein detector to be 861.2 ng / μL. The copy number of the amplified product was calculated according to the formula to be 3.72×10 12 The standard was diluted 10 times in series, with a total of 7 concentrations. The diluted standard was used as a template and TaqMan Real-time PCR amplification was performed using specific primers and probes for human β-actin. A standard curve was drawn, such as Figure 6 A shows: R of the standard curve of the internal reference gene β-actin in human macrophages 2 The amplification efficiency is 0.9994, the slope is -3.3598, and the amplification efficiency is calculated according to the formula E=-1+10(-1 / slope)=98.44%.

[0083] After treating mouse mononuclear macrophage leukemia cells RAW264.7 with PBS, the total DNA of the cells was extracted as a template to amplify the mouse internal reference gene β-actin. The DNA concentration was measured by NanoDrop 2000 micro-nucleic acid protein detector to be 867.9 ng / μL. The copy number of the amplified product was calculated according to the formula to be 4.28×10 12 The standard was diluted 10 times in series, with a total of 7 concentrations. The diluted standard was used as a template and TaqMan Real-time PCR amplification was performed using specific primers and probes for mouse β-actin. A standard curve was drawn, such as Figure 6 Figure B: R of the standard curve of the internal reference gene β-actin in mouse macrophages 2 The value is 0.9968, the slope is -3.1937, and the amplification efficiency is calculated according to the formula E=-1+10(-1 / slope)=105.64%.

[0084] The amplification efficiency E of the two standard curves was in the range of 90-110%, and R 2 They were also greater than 0.98, indicating that the quality of the standards was good and the standard curve had high credibility, and could be used for subsequent Real-time PCR absolute quantification experiments.

[0085] Experimental Example 3 Absolute quantitative qPCR technology after optimization of conditions to determine the intracellular Mtb load in Mtb-infected human (THP-1 induced to macrophages by phorbol ester) and mouse macrophages (RAW264.7)

[0086] 1. TaqMan probe qPCR absolute quantitative determination of intracellular Mtb load

[0087] 1.1 Perform TaqMan Real-time PCR reaction

[0088] When detecting the intracellular Mtb load, the template of the TaqMan Real-time PCR reaction is the total DNA extracted from the cells of the Mtb-infected experimental group. The probe of the tuberculosis-specific insertion sequence IS986 carries a FAM fluorescent group, while the probe of the internal reference gene β-actin carries a ROX fluorescent group. The emission and reception spectra of these two fluorescent groups are different, so the content of the tuberculosis-specific insertion sequence IS986 and the internal reference gene β-actin can be detected simultaneously in the same PCR reaction system. The primers and probes of the TaqMan Real-time PCR reaction are selected according to the cell source. For human cells, the primers and probes of the tuberculosis-specific insertion sequence IS986 and the human internal reference gene β-actin are selected, while for mouse cells, the primers and probes of the tuberculosis-specific insertion sequence IS986 and the mouse internal reference gene β-actin are selected. The TaqMan Real-time PCR reaction system is shown in Table 7. Four replicate wells are set for each sample.

[0089] Table 7 TaqMan Real-time PCR reaction system

[0090]

[0091] 1.2 Calculation of intracellular Mtb load

[0092] After TaqMan Real-time PCR amplification, the tuberculosis load in the sample is calculated based on the calculated copy numbers of the tuberculosis IS986 gene and the human / mouse β-actin gene contained in each total DNA sample. The calculation formula is as follows: tuberculosis load = tuberculosis IS986 gene copy number / human / mouse β-actin gene copy number

[0093] Experimental results: Based on the standard curve obtained by the above TaqMan Real-time PCR amplification and the Ct values ​​of the human / mouse β-actin gene and the Mtb IS986 gene in the sample to be tested, the copy numbers of the β-actin gene and the Mtb IS986 gene in the co-culture system of human macrophages and Mtb, and the copy numbers of the β-actin gene and the Mtb IS986 gene in the co-culture system of mouse macrophages and Mtb were calculated respectively, and then the formula: Mtb load = Mtb IS986 gene copy number / β-actin gene copy number was used to calculate the intracellular Mtb load in the sample to be tested for analysis. Figure 7 As shown: Whether it is the co-culture system of human macrophages and Mtb ( Figure 7 A), or the co-culture system of mouse macrophages and Mtb ( Figure 7 In B), there was no statistical difference in the intracellular Mtb load at 6-24h. Compared with these three time points, the intracellular Mtb load increased at 48h, and the difference was statistically significant (p<0.01). It is known that the reproduction cycle of Mtb is about 18 to 24 hours. The results suggest that Mtb continues to reproduce over time in human and mouse macrophages. TaqMan probe qPCR can absolutely measure the intracellular Mtb load.

[0094] Experimental Example 4 Optimization of total DNA extraction method

[0095] Since Mycobacterium tuberculosis has a thick cell wall and a high lipid content, the effect of extracting the bacterial DNA using a conventional bacterial DNA extraction kit is not good. This experiment compared the concentration and quality of total DNA extracted by the Trizol method and the glass bead method, and ultimately found that the quality of total DNA extracted by the glass bead method was higher and was conducive to the subsequent determination of Mtb load.

[0096] Total DNA extraction in Mtb and macrophage co-culture system (Trizol method):

[0097] ① Taking the six-well plate cell culture as an example, first collect the cell samples of Mtb and macrophage co-culture, add 1mL Trizol reagent to each well, incubate on ice for 5 minutes, repeatedly blow and wash with a pipette tip to collect the cell samples, and place them in a sterile 1.5mL centrifuge tube;

[0098] ② Add 400uL chloroform to each tube, mix well and let stand at room temperature for 15min, centrifuge at 12000rpm for 10min, after the reagents are separated, transfer the upper aqueous phase to a new sterile centrifuge tube for RNA extraction, then use 500uL anhydrous ethanol to precipitate the DNA in the middle layer and organic phase, and let stand at room temperature for 3min;

[0099] ③ Centrifuge at 2000×g for 5 min, remove the supernatant and collect the DNA precipitate;

[0100] ④ Wash the DNA precipitate with 1 mL of 0.1 M sodium citrate containing 10% ethanol, place at room temperature for 30 min, centrifuge at 2000 × g for 5 min at 4°C, discard the supernatant, and repeat the wash once more;

[0101] ⑤ Wash the DNA precipitate with 1 mL of 75% ethanol, place at room temperature for 10-20 min (invert and mix during this period), centrifuge at 2000×g at 4°C for 5 min, and discard the supernatant;

[0102] ⑥ Leave the DNA to dry at room temperature for about 5 to 15 minutes, and dissolve the DNA with an appropriate amount of RNase-Free water (about 30-50uL, depending on the amount of DNA precipitation).

[0103] Total DNA extraction in Mtb and macrophage co-culture system (glass bead method):

[0104] Use a bacterial DNA extraction kit (OMEGA-D3350) to extract the total DNA of bacteria and cells in the co-cultured cell samples. Turn on the water bath and adjust the water temperature to 37°C, 55°C, and 65°C for use. The specific steps for extracting DNA are as follows:

[0105] (1) Collect Mtb-infected cell samples, add 100 μL sterile water to resuspend, then add 10 μL Lysozyme and incubate at 37°C in a water bath for 30 min;

[0106] (2) Weigh 25 mg of glass beads and add them to each centrifuge tube. Vortex at high speed for 5 min at room temperature. Allow the beads to settle and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0107] (3) Add 100 μL BTL buffer and 20 μL proteinase K solution and vortex to mix. Place the sample in a 55°C water bath for 1 hour to completely lyse (take out and vortex every 20-30 minutes);

[0108] (4) Add 5 μL RNase A and mix by repeated inversion, and incubate at room temperature for 5 min;

[0109] (5) Centrifuge at 10,000 × g for 2 min at room temperature and carefully transfer the supernatant to a new centrifuge tube, avoiding aspirating the precipitate.

[0110] (6) Add 220 μL BDL buffer, vortex to mix, and place in a 65°C water bath for 10 min;

[0111] (7) Add 220 μL of anhydrous ethanol and vortex at high speed for 20 seconds. If a precipitate appears at this time, repeatedly blow it 10 times with the pipette tip to break up the precipitate;

[0112] (8) The DNA Mini Binding Column was placed on a 2 mL collection tube, and all the solution obtained in step (7) was transferred into the column. The column was centrifuged at 10,000 × g for 1 min at room temperature, and the collection tube and filtrate were removed.

[0113] (9) Place the binding column on a new 2 mL collection tube, add 500 μL of HBC buffer, centrifuge at 10,000 × g at room temperature for 1 min, and remove the filtrate;

[0114] (10) Place the binding column on the same 2 mL collection tube, add 700 μL of DNA elution buffer, centrifuge at 10,000 × g for 1 min at room temperature, and remove the filtrate;

[0115] (11) Repeat step (10);

[0116] (12) Place the binding column in the same 2 mL collection tube and centrifuge at 10,000 × g for 2 min at room temperature to dry the matrix of the binding column;

[0117] (13) Place the binding column on a 1.5 mL centrifuge tube, add 50 μL of sterile water preheated to 65°C to dissolve the DNA, and place at room temperature for 3-5 minutes;

[0118] (14) Centrifuge at 10,000 × g for 1 min at room temperature to elute genomic DNA;

[0119] (15) Repeat steps (13) and (14), add sterile water preheated to 65°C, and perform a second elution to increase the yield.

[0120] The concentration and purity of the extracted DNA were detected using Nanodrop 2000 micro-nucleic acid protein detector, and PCR amplification was performed to detect the concentration and purity of the PCR amplification product. The PCR amplification system is shown in Table 2.

[0121] Experimental results: After human monocytic leukemia THP-1 cells were induced into macrophages by phorbol ester PMA, macrophages were infected at a concentration ratio of cell number: Mtb number = 1:10, and PBS was used as a solvent control for Mtb bacterial solution. Then, the total DNA in the co-culture system of Mtb and macrophages was extracted by two methods (Trizol method and glass bead method), and the quality of the total DNA extracted by the two methods was identified by the total DNA concentration and purity as well as the PCR product concentration. The results are shown in Tables 8 and 9: The total DNA extracted by the two methods has a higher purity in terms of purity, although the extracted DNA concentration is relatively low. Subsequently, we used the total DNA as a template for PCR amplification of the internal reference gene β-actin and the Mtb specific gene IS986. The reagents and volumes added to the reaction system are shown in Table 2, and the amplification conditions of the PCR reaction are shown in Table 3, Tm = 64°C. The concentration and purity of the PCR product were determined.

[0122] Table 8 Determination of total DNA concentration and purity extracted by glass bead method

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[0124] Table 9 Total DNA concentration and purity determination extracted by Trizol method

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[0126] From Tables 10 and 11, it can be seen that the concentration and purity of the PCR products amplified as templates are roughly the same for total DNA extracted by the glass bead method or the Trizol method. In summary, the concentration and quality of the products amplified by PCR using the same two primers for total DNA extracted by the two methods are roughly the same. Although the concentration of total DNA extracted by the glass bead method is slightly lower, it does not affect subsequent use. In addition, when the total DNA extracted by the Trizol method is dissolved in RNase-Free water, the precipitate cannot be completely dissolved (e.g. Figure 8 As shown in the figure, this will result in the inability to accurately measure the total DNA as the template DNA in the reaction system when the Mtb load is detected later. Therefore, considering the stability and accuracy of the DNA concentration in the subsequent Mtb load measurement experiment, it is more appropriate to use the glass bead method to extract the total DNA in the cell and bacterial co-culture system.

[0127] Table 10 Determination of product concentration and purity of PCR amplification using total DNA extracted by glass bead method as template

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[0129]

[0130] Table 11 Determination of product concentration and purity of PCR amplification using total DNA extracted by Trizol as template

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[0132] In summary, this technical solution optimizes and innovates the absolute quantitative qPCR technology, designs dual fluorescent probes, respectively labels the specific gene and internal reference gene of Mtb, and then uses the absolute quantitative qPCR technology to simultaneously determine the bacterial gene copy number and the internal reference gene copy number in the co-culture system of Mtb and macrophages. Through the calculation formula (Mtb load = Mtb IS986 gene copy number / internal reference gene β-actin copy number), the Mtb load per unit cell can be accurately obtained. This optimized technology is not only innovative, but also can be used as a direct indicator to evaluate the anti-tuberculosis effect, and has broad prospects for promotion and application in scientific research and even clinical diagnosis of tuberculosis treatment.

[0133] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A primer for detecting Mycobacterium tuberculosis based on an internal reference gene and a bacterial gene, characterized in that: The primers include a bacteria-specific insertion sequence IS986 primer and an internal reference gene β-actin primer. The nucleotide sequence of the bacteria-specific insertion sequence IS986 primer is shown in SEQ ID NO: 1, and the nucleotide sequences of the internal reference gene β-actin primer are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. A probe for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes, characterized in that: The probe comprises a bacteria-specific insertion sequence IS986 probe and an internal reference gene β-actin probe. The nucleotide sequence of the bacteria-specific insertion sequence IS986 probe is shown in SEQ ID NO:4, and the nucleotide sequence of the internal reference gene β-actin probe is shown in SEQ ID NO:5 and SEQ ID NO:

6.

3. A probe for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes according to claim 2, characterized in that: The 5' end of the probe is labeled with a fluorescent group, the 3' end of the probe is labeled with a quenching group, the fluorescent group of the internal reference gene β-actin probe is red fluorescent ROX, the fluorescent group of the bacteria specific insertion sequence IS986 probe is green fluorescent FAM; the quenching group is BHQ.

4. A kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes, characterized in that: The method comprises the primer according to claim 1 and the probe according to claim 2.

5. A kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes according to claim 4, characterized in that: Before use, the total DNA in the co-culture system of Mtb and macrophages was extracted using the glass bead method.

6. A kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes according to claim 5, characterized in that: When the kit is used to detect the Mtb load, the Mtb load = the Mtb IS986 bacterial gene copy number / the internal reference gene β-actin copy number.

7. A kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes according to claim 6, characterized in that: The technical formula for copy number is: copy number (copies / μL) = DNA concentration (ng / μL) × 6.02 × 10 23 ×10 -9 / DNA relative molecular mass, where DNA relative molecular mass = number of base pairs in DNA fragment × 660.

8. A kit for detecting Mycobacterium tuberculosis based on internal reference genes and bacterial genes according to claim 7, characterized in that: The Tm of the gradient PCR reaction of primers for the bacterial specific insertion sequence IS986 in the co-culture system of Mtb and macrophages was 52.5-64℃.

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